Synthesis and growth regulatory activity of a prototype member of a new family of aminothiol radioprotectors

ABSTRACT

The synthesis, growth inhibition and radioprotective activity of the PrC-210 aminothiol, 3-(methyl-amino)-2-((methylamino)methyl)propane-1-thiol, and its polyamine and thiolated polyamine progenitors are reported. All of the molecules significantly inhibited growth of cultured normal human fibroblasts. The combination of an ROS-scavenging thiol group and a positively charged alkyl-amine backbone provided the most radioprotective aminothiol molecule.

This application is a continuation of U.S. patent application Ser. No.13/957,188, filed Aug. 1, 2013, which claims priority to U.S. patentapplication Ser. No. 61/713,050, filed Oct. 12, 2012, all of which areincorporated herein by reference in their entirety.

FIELD OF INVENTION

Provided herein is technology related to the synthesis, growthinhibition, and radioprotective activity of aminothiol compounds, e.g.,3-(methyl-amino)-2-((methylamino)methyl)propane-1-thiol (PrC-210 and theanalog PrC-211), and related polyamine compounds, thiolated polyaminecompounds, and progenitors. All of the molecules significantly inhibitedgrowth of cultured normal human fibroblasts. The combination of anROS-scavenging thiol group and a positively charged alkyl-amine backboneprovided the most radioprotective aminothiol molecule.

BACKGROUND OF THE INVENTION

Radiotherapy-induced dermatitis is a common side effect seen in up to85% of patients who receive a course of radiotherapy as part of theircancer therapy regimen. A topically administered radioprotector thatcould be applied prior to radiotherapy on each of the 30 irradiationdays would reduce pain and long term scarring and would improve patientcompliance in receiving all days of treatment. Skipped radiotherapy dayshave a discernible risk for the patient as measured by a decrease in 5year survival rate for breast cancer.

OBJECTS AND SUMMARY OF THE INVENTION

There is also a need for new systemically administered radioprotectors(see, e.g., Examples 2, 3, 4, 6, and 7) that lack the side effects ofnausea/vomiting (see, e.g., Example 4) and hypotension/fainting (see,e.g., Example 5) that have hampered the use of current generationaminothiol radioprotectors, most notably the five carbonaminothiophosphonate, amifostine. The aminothiol radioprotector designconcepts of: (i) a flexible alkyl chain backbone, which carries apositive charge due to one or more amine groups, to achieve ionicinteraction and concentration around negatively charged DNA in cells,and (ii) the presence of a free or capped thiol group to scavenge oxygenfree radicals formed from ionizing radiation, have been used before inprograms to build radioprotective molecules within both the U.S. and theformer Soviet Union.

In the present investigation, we disclose a process in which: (i) thenumber of alkylamine segments in the aminothiol backbone issystematically increased to increase drug-DNA affinity and ionicinteraction, resulting in increased growth inhibition that is associatedwith this enhanced drug-DNA interaction, and (ii) the placement or‘display’ of a free thiol reactive oxygen species (ROS) scavenger at theend of a short alkyl side chain that displaces or ‘displays’ thescavenger moiety away from the DNA backbone to theoretically enable ROSscavenging before ROS attack on dG bases within cellular DNA (see, e.g.,FIG. 3 and Example 7). This work has resulted in a small family of newaminothiol molecules, the prototype of which, PrC-210, is described ininitial detail here.

The synthesis of PrC-210, shown in Scheme 1, started with a doubledisplacement of chloride from 1 using N-methyl mesitylene-sulfonamide(2) and sodium hydride, to form allylic sulfonamide 3. Hydroboration of3 afforded clean conversion to sulfonamide alcohol 4. Using standardconditions, 4 was converted to mesylate 5 which was immediately treatedwith potassium thioacetate to form 6. Following an establishedprocedure, the mesitylene (Mts) protecting groups were removed withHBr/HOAc, in the presence of excess phenol. The deblocking procedurealso hydrolyzed the thioacetate group. Work up resulted in a mixture ofPrC-210 and the corresponding disulfide (dimer). The mixture was treatedwith 2-mercaptoethanol to cleave the disulfide and the product, PrC-210,was precipitated from EtOH as the HCL salt. Subsequentrecrystallizations removed the sulfurous odor (see, e.g., Example 1).

The synthesis of PrC-211, shown in Scheme 2, employed a modification ofthe route used for PrC-210. An attempt was made to form sulfonamide 10directly by displacement of chloride from 1 using mesitylenesulfonamide,activated with sodium hydride. A complex mixture formed, from which 10could not be isolated in pure form. Alternatively, 1 was treated withpotassium phthalimide to form allylic phthalimide 8. Removal of thephthalate groups with hydrazine provided 9, which upon treatment withmesitylenesulfonyl chloride afforded the bis-sulfonamide 10 in goodyield. Using the sequence from the PrC-210 preparation, hydroboration,mesylation, thioacetate displacement and deblocking, PrC-211 wasobtained as the HCl salt and subsequently recrystallized.

The amine side chains, synthesized according to the route illustrated inScheme 3, were constructed as sulfonamide-protected intermediates, eachwith a single point of attachment (N—H), at one terminus, for couplingto the olefinic core (FIG. 1E). Preparations for sulfonamides 17 and 20have previously been described. A convenient alternative approach wasfound that employed a modification of a reported method, starting withN-(4-bromobutyl)phthalimide (14). Displacement with N-ethylmesitylenesulfonamide and sodium hydride, afforded 15, which upontreatment with hydrazine, was converted to 16. Amidation withmesitylenesulfonyl chloride, under standard conditions, gave 17.Sulfonamide 17, a protected diamine, represents the smallest amine sidechain unit in the series, which includes 20, 21, and 22. Sulfonamide 17was systematically elaborated to the subsequent side chain units usingthe same methodology as described for 17.

Coupling of the various amine side chains to the olefinic core is shownin Scheme 4. The synthesis commenced with the known TBS-protectedallylic alcohol 23. Mesylation, using standard conditions, provided theactivated intermediate 24. Coupling with a sulfonamide side chain andsodium hydride provided 25. Removal of the TBS-protecting groups withHCl afforded diol 26. Treatment of 26 with 1 equiv of benzoyl chloride,in the presence of pyridine, afforded alcohol 27 as a mixture ofisomers. Activation of the allylic alcohol 27, for amine side chaincoupling, was attempted with methanesulfonyl chloride, resulting in acomplex mixture. Alternatively, the alcohol was converted to bromide 28,with phosphorous tribromide, and immediately coupled with a second sidechain unit to form 29. Hydrolysis of the benzoate group was carried outunder standard conditions to afford the versatile polyamine alcoholintermediate 30, which was a common intermediate for the formation ofboth polyamines and polyamine thiols.

Conversion of the versatile intermediate 30 to polyamines and polyaminethiols is shown in Scheme 5. Treatment of 30 with methanesulfonylchloride and triethylamine afforded the mesylate 31. Displacement withN-ethyl mesitylenesulfonamide, activated with sodium hydride, provided32 which was deblocked with HBr/HOAc and phenol, to afford crudepolyamine 33. Free-basing with potassium carbonate followed by HCltreatment gave 33 hydrochloride salt as a mixture of cis/trans isomers.Mesylate intermediate 31 was treated with potassium thioacetate to form34 which was de-blocked, using the same method as for 32, to form 35.Free-basing and treatment with HCl provided 35 as the hydrochloridesalt.

FIG. 1A provides a summary of the first set of polyamine structures thatwas synthesized using the strategies outlined in Schemes 3-5. Weanticipated that these drug molecules would provide radio- andchemo-protection to human cells by inducing a cell cycle block inmammalian cells at the G1/S cell cycle border because of tight bindingof the (+) charged polyamine backbones to the (−) charged DNA backbone.Such a block can provide time for DNA repair in radiation- ormutagen-treated cells before washout of the polyamine drug andrestoration of cell cycle progression.

FIG. 1B shows a nearly identical set of polyamines now with the additionof a thiol group to each molecule. The goal in synthesizing thesemolecules was to achieve the same cell cycle inhibition anticipated forthe FIG. 1A molecules, but now combined with the addition of a thiolgroup that could serve as a scavenger for the burst of short-lived ROSthat is generated when mammalian tissue is irradiated. The aminothioland polyamine structures in FIG. 1B are growth inhibitory when added torapidly growing, normal human fibroblasts, and the potency of growthinhibition was directly related to the number of (NH—(CH₂)_(n)) segmentspresent within the molecule (FIG. 1D).

Early in vitro studies of radioprotection with cultured cellsdemonstrated that the long, polyamine structures were sogrowth-inhibitory (FIG. 1C) that we could not add sufficient moles ofthe thiolated polyamines (e.g., PrC-117) to cell cultures that wouldenable the thiol groups to significantly scavenge and radioprotect whenthe tissue culture cells were irradiated. This provided the impetus todesign and synthesize the PrC-200 series of small aminothiols, of whichPrC-210 is the prototype.

To determine if PrC-210 could function as a topical radioprotector thatcould prevent radiation dermatitis when applied prior to a cancerpatient's daily radiotherapy, a rat assay that realistically mimicshuman radiation dermatitis was created. This assay quantifies theseverity of radiation dermatitis in rat skin 13 days after a singleradiation dose of 17.33 Gy. In this study, PrC-210 was applied to ratskin in an ethanol/water delivery vehicle four times in the 2 h beforeirradiation, and control rats received only topical vehicle beforeirradiation (FIG. 2). Additional drug/irradiation groups of three ratseach received either the alkyl-thiol moiety or the alkyldiamine moietyfrom PrC-210 in the same delivery vehicle. Following the 17.33 Gyirradiation of a 1.5-3.0 cm patch of dorsal skin on the rats' backs,rats were scored 13 days later. The ability of the PrC-210 aminothiol tocompletely prevent radiation dermatitis was striking. Otherradiodermatitis test groups exploring efficacy of thiolated polyaminesor other PrC-200 series aminothiols have shown that PrC-210 is the mostpotent and most effective topical aminothiol to date.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presenttechnology will become better understood with regard to the followingdrawings:

FIGS. 1A-1E show growth inhibition of normal human cells in culture bypolyamine and aminothiol molecules. FIG. 1A shows structures of firstgeneration synthetic polyamines. FIG. 1B shows first generation‘thiolated polyamines’ and the next generation aminothiol, PrC-210. FIG.1C shows the addition of molecules at indicated concentrations to logphase, growing cultures of diploid human fibroblasts and the cellnumbers measured 4 days later. FIG. 1D shows that growth inhibition isdirectly related to the number of [NH—(CH₂)] segments present in thepolyamine or aminothiol molecule. FIG. 1E shows the olefinic core commonto polyamine and polyamine-thiol series.

FIG. 2 shows the prevention of radiation-induced dermatitis in rat modelby prior topical application of the PrC-210 aminothiol or its componentstructures. Molecules were applied to skin in an alcohol/water deliveryvehicle prior to receiving a 17.33 Gy radiation dose to the 1.5 cm×3.0cm skin site. Radiation dermatitis severity (e.g., % of the site coveredby scab material) was scored 13 days following irradiation.

FIG. 3 shows a schematic for the contemplated mechanism of protectionconferred by PrC-210 to DNA against reactive oxygen species.

FIG. 4 shows the systemic radioprotective effect of PrC-210 inpre-exposure mouse models.

FIG. 5 shows that orally administered PrC-210 confers 100% protectionagainst an otherwise 100% lethal dose of whole-body radiation.

FIG. 6 shows that orally administered PrC-210 can be administered 60minutes before irradiation to confer 100% protection against anotherwise 100% lethal dose of whole-body radiation.

FIG. 7A shows that amifostine at a maximum human dose equivalent inducesretch and emesis in a ferret model (4 ferrets). FIG. 7B shows thatPrC-210 at a likely maximum human dose equivalent does not induceretch/emesis in a ferret model (10 ferrets).

FIGS. 8A-8D show that PrC-210 does not cause hypotension side effects.FIG. 8A shows the drugs and dosages used for the study. FIG. 8B showsrecorded blood pressure after administration of amifostine. FIG. 8Cshows recorded blood pressure after administration of PrC-210. FIG. 8Dshows a comparison of the changes in blood pressure caused by controls(saline and epinephrine), amifostine, and PrC-210.

It is to be understood that the figures are not necessarily drawn toscale, nor are the objects in the figures necessarily drawn to scale inrelationship to one another. The figures are depictions that areintended to bring clarity and understanding to various embodiments ofapparatuses, systems, and methods disclosed herein. Wherever possible,the same reference numbers will be used throughout the drawings to referto the same or like parts. Moreover, it should be appreciated that thedrawings are not intended to limit the scope of the present teachings inany way.

EXAMPLES Example 1

During the development of embodiments of the technology provided herein,experiments were conducted demonstrating that embodiments of thecompounds provided (e.g., PrC-210) lacked noxious odors (e.g., sulfurousodors) associated with conventional compounds and technologies. Subjectswere exposed to a solution comprising PrC-210 at the upper limit of whatan approximate single human dose of PrC-210 is contemplated to be and adilution series of 2-mercaptoethanol (2-ME). Each subject assigned a“smell score” to the PrC-210 by comparing the smell of the PrC-210 withthe 2-ME dilutions; the smell score denotes the 2-ME dilution having asulfurous thiol smell that most closely matched the sulfurous thiolsmell of the single human dose of PrC-210. One subject assigned a smellscore of 8 and the other subject assigned a smell score of 7,corresponding to 1:18,750 and 1:93,750 dilutions of 2-ME. These resultsshow that PrC-210 at a concentration of approximately a single maximumhuman dose has a thiol odor that is 56,250-fold lower than 2-ME (e.g.,93,750-18,750=75,000; 75,000÷2=37,500; 37,500+18,750=56,250). A56.250-fold dilution of 2-ME is nearly odor free.

Example 2

During the development of embodiments of the technology provided herein,experiments were performed demonstrating that administering anintraperitoneal, systemic dose of PrC-210 to a mouse at a time 30minutes before exposure to 8.75 Gy of whole-body radiation confers 100%survival (FIG. 4, “IP PrC-210”). Exposure of a control mouse to the samedose of radiation in the absence of PrC-210 administration (e.g., awater-only control) is 100% lethal (FIG. 4, “IP Water”). Each treatmentgroup contained 20 mice. The dose of PrC-210 was 252 μg/g body weight.

Example 3

During the development of embodiments of the technology provided herein,experiments were performed demonstrating that PrC-210 administeredorally confers 100% protection against an otherwise 100% lethal dose ofwhole-body radiation. Female Sprague-Dawley rats received 200 μl dosesof PrC-210 dissolved in water by gavage a time of 60 minutes prior toirradiation (time=−60 min). At time=0 min, rats were irradiated with 9.0Gy of whole-body radiation in a ¹³⁷Cs irradiator. Rats were returned tohousing and their weights and survival were monitored for the next 60days. There were six rats in each of the treatment groups. As shown byFIG. 5, oral PrC-210 confers 100% protection against an otherwise 100%lethal dose of whole-body radiation.

In additional experiments, female ICR mice received 200 μl doses (e.g.,900 μg/g body weight) of PrC-210 dissolved in water by gavage at theindicated times prior to irradiation (See FIG. 6). At 0 min, mice wereirradiated with 9.0 Gy of whole-body radiation in a CS 137 irradiator.Mice were returned to housing and their weights and survival weremonitored for the next 60 days. There were six mice in each of thetreatment groups. As shown in FIG. 6, the oral dose can be administered60 minutes before irradiation and confer this effect.

Example 4

During the development of embodiments of the technology provided herein,experiments were performed demonstrating that a systemic PrC-210 dose,at the ferret equivalent of the likely highest systemic human dose thatwould be used, caused zero retching or emesis responses when tested in10 ferrets (see FIG. 7B). Amifostine, at the ferret equivalent of thehighest human amifostine dose used, caused retch/emesis responses ineach of the four ferrets tested (FIG. 7A). Loperamide, a positivecontrol for retch/emesis response, caused robust retch/emesis responsein each of the 14 ferrets when administered 14 days after the singlePrC-210 or amifostine test of the ferret.

Example 5

During the development of embodiments of the technology provided herein,experiments were performed demonstrating that PrC-210 does not causehypotension side effects caused by conventional radioprotectors (seeFIGS. 8A-8D). Blood pressure was monitored in the aortic artery of ratsusing a high-fidelity pressure transducer. Rats were continuallyanesthetized with 2% isofluorane, and drug doses were administered byintraperitoneal injection. Rats received intraperitoneal salineinjections until these injections induced no change in monitored bloodpressure. Indicated doses of PrC-210 or amifostine (FIGS. 8A-8D) wereadministered and changes in blood pressure and heart rate were recorded.A condensed summary of drug-induced changes for all cannulated rats isshown in FIG. 8C. Doses of epinephrine (Epi; hypertensive) were alsorecurrently administered to test if previous PrC-210 or amifostine dosescould be modified.

Example 6

During the development of embodiments of the technology provided herein,experiments were conducted to test the biodistribution of an intravenousbolus dose of radiolabeled (e.g., ¹⁴C) PrC-210 in a rat host. In theseexperiments, an intravenous bolus of 100 μCi of ¹⁴C PrC-210 wasadministered into an indwelling catheter in a 300 g rat. Distributiondata from this experiment are shown in Table 1:

TABLE 1 ¹⁴C PrC-210 Dose C_(max) T_(max) Recovery 1 Recovery Recovery 2Recovery 3 Group (Route) (ng-eq/g) (hr) (excreta) (carcass) (systemic)(total) 1 100 μCi (IV) 21,400 0.5 74.34% 6.89% 81.23% 84.20% Recovery 1(excreta) = urine + urine wipes + feces; Recovery 2 (systemic) =Recovery (excreta) + Recovery (carcass) Recovery 3 (total) = Recovery(systemic) + Recovery (skin) + cage wash + cage wipe + enclosure

After the intravenous administration of ¹⁴C PrC-210 the main route ofelimination was via urine, accounting for approximately 50% of theadministered radioactivity; feces accounted for approximately 10%.

Example 7

During the development of embodiments of the technology provided herein,embodiments of compounds described herein and/or encompassed by thesynthetic schemes were tested for systemic toxicity and radioprotectionin mouse and rat models (Table 2). Toxic doses are shown as the “maximumtolerated dose” (“MTD”) for intraperitoneal (IP), oral, and subcutaneous(SC) administration routes in Table 2.

To measure “% Survival” as a radioprotection scored endpoint, groups of10-20 mice received the indicated drug, at the indicated dose, via theindicated delivery route, and were then exposed to a whole-bodyradiation dose (8.75-9.0 Gy) that killed 100% of the mice in the groupthat received only delivery vehicle and radiation. Survival ratesgreater than the 0% Survival seen in the vehicle control group wereattributed to systemic radioprotection conferred by the systemicallyadministered radioprotector molecule.

TABLE 2 (3) (1) (2) Mouse (4) (5) Mouse Rat ORAL Rat Mouse (6) (7)Molecule IP IP MTD ORAL SC Mouse Rat Name MW MTD MTD (ug/g b.w.) MTD MTD% Survival^(e) % Survival^(f) PrC-210 148  504^(a) 485 1780 1974 431100% (at IP 0.5 MTD: 100% (at ORAL 0.5  422^(b) 252 μg/g) MTD: 900 μg/g)98% (at IP 0.5 MTD: 211 μg/g) 100% (at ORAL 0.87 MTD: 1550 μg/g)^(g)PrC-210 294 155 — — — — 37% (at IP 0.9 MTD: — Disulfide 140 μg/g) <5%(at IP 0.5 MTD: 78 μg/g) PrC-211 120 475 — — — — 100% (at IP 0.9 MTD: —427 μg/g) 47% (at IP 0.5 MTD: 238 μg/g) 37% (at IP 0.25 MTD: 118 μg/g)PrC-301 162 625 — — — — <5% (at IP 0.5 MTD: — 312 μg/g) PrC-303 2741860  — — — — ND^(h) — PrC-304 188 1340  — — — — <5% (at IP 0.5 MTD: —670 μg/g) Prc-307 176 166 — — — — 12% (at IP 0.5 MTD: — 83 μg/g)Amifostine 214  800^(c) — — — — 88% (at IP 0.5 MTD: —  760^(d) 400 μg/g)^(a)Determined using probit analysis of survival data ^(b)Determinedusing best fit analysis of survival data ^(c)Published value^(d)Experimentally determined ^(e)8.63 Gy whole-body radiation ^(f)9.5Gy whole-body radiation ^(g)This dose was the only one tested ^(h)notdetermined

As shown by Table 2, aminothiols (e.g., PrC-210 and PrC-211) providedradioprotection when administered via intraperitoneal and/or oralroutes. In particular, PrC-210 and PrC-211 conferred radioprotection atdoses less than the MTD.

Doses expressed as a fraction of “MTD” are expressed as a fraction ofthe “maximum tolerated dose”. Doses expressed in units of μg/gcorrespond to micrograms of the compound per gram of body weight.

1. A method for protecting a subject from ionizing radiation, the methodcomprising administering systemically to the subject a radioprotectorcompound comprising a free thiol and a positively-charged backbone viaintraperitoneal injection intravenous injection, or enterally by mouthin an amount effective to protect the subject from ionizing radiation.2. The method of claim 1 wherein the positively-charged backbone of theradioprotector compound comprises an amine.
 3. The method of claim 1wherein the radioprotector compound comprises a structure according to:

wherein R and R′ are independently selected from H, CH₃, alkyl, andheteroalkyl.
 4. The method of claim 1 wherein administering systemicallyto the subject the radioprotector compound does not cause a side effectof nausea, vomiting, hypotension, or fainting in the subject.
 5. Themethod of claim 1 wherein the amount administered is effective to blockcell cycle progression at the G1/S cell cycle border.
 6. The method ofclaim 1 wherein the radioprotector compound is sulfurous odor-free. 7.The method of claim 1 wherein the amount administered is effective toinhibit the growth of a cell.
 8. The method of claim 1 wherein theamount administered is effective to permit restoration of cell cycleprogression.
 9. The method of claim 1 wherein the administering iseffective to bind the positively-charged backbone to a DNA whiledisplaying the free thiol away from the DNA.
 10. The method of claim 1wherein the administering is effective to scavenge reactive oxygenspecies.
 11. The method of claim 1 wherein the subject is a mammal. 12.The method of claim 1 wherein the subject is a human.
 13. The method ofclaim 1 wherein the subject is a human comprising a cell exposed toradiation for medical purposes.
 14. The method of claim 1 wherein aneffective amount of the radioprotector compound is administeredsystemically at an effective time before or after radiation exposure.15. A method of providing protection to a subject's DNA against areactive oxygen species, the method comprising: a) administeringsystemically to the subject a radioprotector compound comprising a freethiol and a positively-charged backbone via intraperitoneal injection,intravenous injection, or enterally by mouth in an amount effective toprotect the subject's DNA from reactive oxygen species; b) binding thepositively-charged backbone of the radioprotector compound to a DNA ofthe subject; and c) scavenging a reactive oxygen species with the freethiol, wherein the radioprotector compound provides protection to thesubject's DNA against the reactive oxygen species.
 16. The method ofclaim 15 wherein the radioprotector compound comprises a structureaccording to:

wherein R and R′ are independently selected H and CH₃.
 17. The method ofclaim 16 further comprising inhibiting the growth of a cell.
 18. Amethod of providing radioprotection to a subject, the method comprising:a) providing a radioprotector that lacks the side effects ofnausea/vomiting and hypotension/fainting; and b) administeringsystemically the radioprotector to the subject via intraperitonealinjection, intravenous injection, or enterally by mouth in an amounteffective to provide systemic radioprotection to the subject.
 19. Themethod of claim 18 wherein the radioprotector compound comprises astructure according to:

wherein R and R′ are independently selected from H and CH₃.
 20. Themethod of claim 18 further comprising inhibiting the growth of a cell.21. The method of claim 1 wherein the radioprotector compound issystemically administered enterally by mouth in a manner effective toprovide a systemic radioprotective effect against the ionizingradiation.
 22. The method of claim 1 wherein the radioprotector compoundis systemically administered enterally by mouth in a manner effective toprovide a systemic radioprotective effect against the ionizing radiationthat is equivalent to 20-100% of a systemic radioprotective effectagainst the ionizing radiation provided by intraperitoneal injection ofan amount of the radioprotector compound that is 0.5 times a maximumtolerated dose administered by intraperitoneal injection.
 23. The methodof claim 1 wherein the radioprotector compound is systemicallyadministered enterally by mouth in a manner effective to provide asystemic radioprotective effect against the ionizing radiation that isequivalent to a systemic radioprotective effect against the ionizingradiation provided by intraperitoneal injection of an amount of theradioprotector compound that is 0.5 times a maximum tolerated doseadministered by intraperitoneal injection.
 24. The method of claim 1wherein the amount systemically administered enterally by mouth is anamount of from about 25% to about 61% of a maximum tolerated dosesystemically administered enterally by mouth.